| AimsCisplatin(DDP)has been considered the cytotoxic basis of doublet chemotherapy in lung adenocarcinoma(LUAD).However,many patients will unavoidably face treatment failure due to the emergence of platinum resistance.Currently,the family of fibroblast growth factor receptors(FGFR)have been identified as promising targets for LUAD therapy.Therefore,it is urgent to identify the role and mechanisms of FGFR inhibitors in LUAD,with a view to providing new therapeutic drugs for cisplatin-resistant patients.According to whether covalently binding the P-loop cysteine in the adenosine triphosphate(ATP)pocket of the FGFR intracellular tyrosine kinase domain,FGFR inhibitors are divided into reversible and irreversible FGFR inhibitors.The reversible FGFR inhibitors Erdafitinib and Pemigatinib have been approved by the Food and Drug Administration for the treatment of metastatic urothelial carcinoma(mUC)and cholangiocarcinoma,respectively.The irreversible FGFR inhibitors have become more promising antitumor drugs due to their better binding kinetics and potential ability to overcome the resistance of reversible FGFR inhibitors.FIIN-2,as a representative drug of irreversible FGFR1-4 inhibitors,can inhibit the proliferation of multiple tumor cells,including lung squamous cell carcinoma and large cell carcinoma.However,the therapeutic efficacy of FIIN-2 has not been systematically confirmed in LUAD.Autophagy is an intracellular process that transports damaged,degenerated,or senescent proteins and organelles to lysosomes for degradation,playing a vital role in maintaining cellular metabolism and internal environment stability.Beclin-1,as a central player in autophagy,binds to different molecules such as B cell lymphoma/lewkmia-2(Bcl-2),Vps34 to form the phosphatidylinositol 3-kinase catalytic subunit type 3(PI3KC3)-Beclin-1 complex and plays a role in autophagosome formation and maturation.After autophagy is induced,the cytoplasmic microtubule-associated protein 1 light chain 3(LC3)-I couples with the substrate PE on the membrane surface to form the membrane-bound LC3-II,the hallmark of autophagosome formation.p62 is degraded by the autolysosome through binding to autophagy substrates.Recent studies have proved that autophagy is closely related to tumors,inhibiting or promoting tumor development.Autophagy is mainly triggered by the mammalian target of rapamycin(mTOR)inhibition or the type III phosphatidylinositol 3-kinase(PI3K)complex activation.As the downstream of FGFR,PI3K/AKT is the most classical pathway that mediates mTOR signaling to regulate autophagy.The irreversible FGFR1-4 inhibitor FIIN-2 can inhibit the growth of multiple tumor cells including lung cancer by blocking PI3K/AKT signaling.However,whether the antitumor effect of FIIN-2 is related to autophagy has not been reported yet.In this study,to explore the antitumor effect of the irreversible FGFR inhibitor FIIN-2,we used representative human LUAD cell lines of A549 and cisplatin-resistant A549/DDP.At the same time,the relationship between the anti-LUAD effect of FIIN-2 and autophagy was elaborated.It provides a candidate for LUAD treatment,especially for patients with cisplatin resistance.Methods1.Histopathological types of lung cancer patients were analyzed using hematoxylin-eosin staining(H&E)staining.The expression of FGFR2 in LUAD tissues was analyzed using immunohistochemistry(IHC).The messenger ribonucleic acid(mRNA)expression of FGFR1-4 in A549 and A549/DDP cells was detected by real-time quantitative PCR(RT-qPCR).Subsequently,the expression levels of FGFR2 and its downstream signaling proteins phosphorylate fibroblast growth factor receptor(p-FGFR),FRS2 and phosphorylate FGFR substrate 2(p-FRS2)in the two cells were detected by western blotting.2.The cell counting kit-8(CCK-8)method was used to detect the effect of different concentrations of cisplatin on the viability of A549 and A549/DDP cells,and the IC50 values and resistance index(RI)of cisplatin to the two cells were calculated.Meanwhile,western blot was used to detect the expression of multidrug resistance protein 1(MDR1)in A549 and A549/DDP cells.3.The effects of different concentrations of FIIN-2 on the proliferation of A549 and A549/DDP cells were detected by CCK-8 assay,and the IC50 values were calculated.4.The flow cytometry and Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assays were used to evaluate the effect of FIIN-2 on the apoptotic rates of A549 and A549/DDP cells.The changes of mitochondrial membrane potential(MMP)were detected by JC-1 staining,and western blot was used to detect the effect of different concentrations of FIIN-2 on the key indicators of mitochondrial apoptosis pathway Bcl-2,Bcl-2-associated X protein(Bax)and apoptosis effector protein cleaved Caspase-3.5.Colony formation assay was used to detect the effect of FIIN-2 on the colony-forming ability of A549 and A549/DDP cells.Wound healing assay was used to detect the effect of different concentrations of FIIN-2 on the migration ability of A549 and A549/DDP cells,and western blot was used to detect the effect of FIIN-2 on the expression of migration-related proteins matrix metalloproteinase-2(MMP2)and matrix metalloproteinase-9(MMP9)in A549 and A549/DDP cells.6.To analyze whether FIIN-2 has inhibitory effects on FGFR2 and its downstream signaling,western blot was used to detect the effect of different concentrations of FIIN-2 on the expression of FGFR2 and downstream p-FGFR,p-FRS2,p-AKT and p-ERK.7.Transmission electron microscopy(TEM)and monodansylcadaverine(MDC)method were used to observe the effect of FIIN-2 on the autophagic properties of A549 and A549/DDP cells.Subsequently,western blot was used to detect the effect of FIIN-2 on the expression of autophagy-related proteins Parkin,LC3-II/LC3-I,p62 in A549 and A549/DDP cells.8.Western Blot was used to detect the effect of FIIN-2 on autophagic flux in A549 and A549/DDP cells after chloroquine(CQ)blocked autophagic flux.A549 and A549/DDP cells were transfected with mRFP-GFP-LC3 lentiviral vector to construct cell lines stably expressing mRFP-GFP-LC3.The number of autophagosomes(yellow fluorescence)and autophagolysosomes(red fluorescence)was observed by laser confocal microscopy to further verify the effect of FIIN-2 on autophagic flux.9.To investigate whether FIIN-2 exerted anti-LUAD effects and induced antophagy by inhibiting the mTORC1 signaling,western blot was used to detect the effect of FIIN-2 on p-mTOR.Next,cells were treated with the mTOR agonist MHY1485 alone or in combination with FIIN-2.The cell viability was detected by CCK-8 method,the expression of pro-apoptotic protein Bax,cleaved Caspase-3,and autophagy marker LC3-Ⅱ/LC3-Ⅰ was detected by Western Blot.To explore whether FIIN-2-mediated mTORC1 inhibition is related to the AKT and ERK signaling,AKT agonist SC79 or ERK agonist LM22B-10 were used to treat cells alone or in combination with FIIN-2.Western blot was used to detect the expression of AKT/mTORC1(AKT,p-AKT,mTOR,p-mTOR)and ERK/mTORCl signaling proteins(ERK,p-ERK,mTOR,p-mTOR),respectively.10.Western blot was used to examine the effect of FIIN-2 on Vps34 and Beclin-1 in type Ⅲ PI3K complex.And further the Co-immunoprecipitation(Co-IP)was used to detect the effect of FIIN-2 on Beclin-1 and Vps34.To verify whether FIIN-2induces autophagy by inhibiting mTORCl and further activating the type Ⅲ PI3K pathway,cells were treated with FIIN-2 combined with or without the mTOR agonist MHY1485,and the expression changes of mTOR,p-mTOR,Beclin-1,Vps34,and LC3-Ⅱ/LC3-Ⅰ were detected by western blotting.11.To elucidate the relationship between autophagy and apoptosis caused by FIIN-2,western blot was used to detect the changes of autophagy-and apoptosis-related proteins at different times after FIIN-2 treatment.To further determine whether inhibition of autophagy enhanced FIIN-2-mediated cytotoxicity,cells were treated with the late autophagy inhibitor CQ or early autophagy inhibitor 3-methyladenine(3-MA)alone or in combination with FIIN-2.The cell viability was detected by CCK-8 assay,and the expression levels of pro-apoptotic proteins cleaved Caspase-3,Bax and anti-apoptotic protein Bcl-2 were detected by western blotting.12.The subcutaneous xenograft tumor models of A549/DDP and A549 cells were established,respectively.The nude mice of the two models were divided into four groups and treated with saline solution,CQ,FIIN-2,and CQ combined with FIIN-2.After the mice were sacrificed,tumor tissues were obtained,and the expression of proliferation(Ki67),apoptosis(cleaved Caspase-3)and autophagy(LC3)-related molecules were analyzed by IHC.The TUNEL assay was used to detect apoptosis rate,and western blot detect apoptosis(cleaved Caspase-3),autophagy markers(LC3-II/LC3-I,p62)and p-FRS2 signaling pathway.Results1.FGFR2 is highly expressed in LUADThe results of IHC analysis showed that FGFR2 was expressed in the tissues of 19 patients with LUAD,of which 9(47.37%)had high expression of FGFR2.RT-qPCR results showed that both A549 and A549/DDP cells expressed FGFR1-4 at the mRNA level,and the mRNA level of FGFR2 showed relatively high expression in both cell lines.Western Blot results showed that A549 and A549/DDP cells continuously expressed FGFR2,and the expression levels of FGFR2,p-FGFR,and p-FRS2 in A549/DDP cells were higher than those in A549 cells.2.Identification of cisplatin-resistance of A549/DDP relative to A549 parental cellsThe results of CCK-8 assay showed that the cell viability and IC50 values of A549/DDP cells were significantly higher than those of A549 cells,which were(122.01 ± 28.52)μmol/L and(7.76±3.39)μmol/L,respectively.The RI of A549/DDP cells was 15.7 to cisplatin relative to their parental A549 cell line,and exhibited relatively higher expression of MDR1.3.FIIN-2 inhibited proliferation and induced mitochondria-mediated apoptosis in A549 and A549/DDP cellsThe results of CCK-8 assay showed that FIIN-2 dose-dependently decreased the cell viability of A549 and A549/DDP,and the IC50 value of FIIN-2 on A549/DDP cells was significantly lower than that of A549,which were(16.3±0.4)μmol/L and(31.3±0.2)μmol/L.The results of apoptosis assessment using TUNEL staining and FCM showed FIIN-2 dose-dependently induced the apoptosis of A549 and A549/DDP cells.The results of JC-1 staining to detect MMP showed that the ratio of aggregated JC-1/monomeric JC-1 was reduced after treated with FIIN-2 for 24h.Next,we found that FIIN-2 dose-dependently decreased the level of antiapoptotic protein Bcl-2 and increased the expression of proapoptotic proteins Bax and cleaved Caspase-3 by western blot.Notably,the FIIN-2-induced apoptosis was more pronounced in A549/DDP cells than in A549 cells.4.FIIN-2 inhibited the colony formation and migration ability of A549 and A549/DDP cellsWe observed that FIIN-2 treatment significantly reduced the colony-forming ability of both cells,and the reduction was more evident in A549/DDP cells.Furthermore,the migration ability of A549 and A549/DDP cells was also significantly reduced by treatment with FIIN-2 for 24h or 48h,and the migration-related proteins MMP2 and MMP9 were inhibited.5.FIIN-2 inhibited FGFR2 and its downstream signaling in A549 and A549/DDP cellsThe results of western blot showed that the expressions of FGFR-dependent signaling proteins p-FGFR,p-FRS2,p-AKT and p-ERK in A549 and A549/DDP cells were significantly decreased after treatment with FIIN-2 dose-dependently.In addition,the inhibitory ability of FIIN-2 on p-AKT signal was more obvious in A549/DDP cells than that in A549 cells.6.FIIN-2 increased the autophagic properties of A549 and A549/DDP cellsAs TEM results showed,the number of autolysosomes in A549 and A549/DDP cells increased significantly after treatment with 10μmol/L FIIN-2 for 24h.By using the MDC method,we also observed that FIIN-2 could dose-dependently increase the number of acidic autophagic vesicles in A549 and A549/DDP cells.Next,we found FIIN-2 increased the level of Parkin and the accumulation of LC3-II increased,whereas the expression of p62 decreased.Notably,the above phenomenon was more pronounced in A549/DDP cells than in A549 cells.7.FIIN-2 induced autophagic flux in A549 and A549/DDP cellsTo confirm the effect of FIIN-2 on autophagic flux,CQ was used to prevent autophagolysosome degradation.We found that the combination of CQ further enhanced the LC3-Ⅱ level induced by FIIN-2.Meanwhile,FIIN-2 alone and its combination with CQ reduced p62 expression.Further,the A549 and A549/DDP cell lines stably expressing mRFP-GFP-LC3 were treated with FIIN-2,and the results showed that the number of autophagosomes and autophagolysosomes in the FIIN-2-treated cells was significantly increased.8.FIIN-2 exerted anti-LUAD effect and induced antophagy by inhibiting AKT/ERK-mTORCl signalingThe results showed that FIIN-2 inhibited the expression of p-mTOR(ser2448),indicating that FIIN-2 inhibited the activity of mTORC1,and the inhibitory ability of A549/DDP cells was significantly higher than that of A549 cells.The mTOR agonist MHY1485 could resist the inhibitory cell viability of FIIN-2 and its induced apoptosis and autophagy.The AKT agonist SC79 and the ERK agonist LM22B-10 resisted the inhibitory effect of FIIN-2 on p-AKT and p-ERK,respectively,while upregulating the expression of p-mTOR(ser2448).9.FIIN-2 induced autophagy by inhibiting mTORCl and further activation of the class Ⅲ PI3K pathwayThe results showed that FIIN-2 dose-dependently upregulated the expressions of Beclin-1 and Vps34,especially in A549/DDP cells.The Co-IP results further showed that FIIN-2 promoted the recruitment of more Vps34 by Beclin-1.However,the mTOR agonist MHY1485 could significantly resist the inhibitory effect of FIIN-2 on p-mTOR(Ser2448),and reduce the expression of Beclin-1,Vps34 and the accumulation of LC3-Ⅱ.10.Inhibition of autophagy enhanced FIIN-2-mediated cytotoxicity in A549 and A549/DDP cellsThe results of western blot showed that FIIN-2 treatment of A549 and A549/DDP cells for 6h could upregulate the expression of LC3-Ⅱ and peaked at 24h,accompanied by the down-regulation of p62.Cleaved Caspase-3 and Bax were upregulated with prolonged FIIN-2 treatment(12h in A549 cells,24h in A549/DDP cells),accompanied by the down-regulation of Bcl-2.In addition,neither the early autophagy inhibitor 3-MA nor the late autophagy inhibitor CQ alone could affect the viability of A549 and A549/DDP cells.However,FIIN-2 combined with CQ or 3-MA significantly increased the inhibitory effect of FIIN-2 on cell viability.More importantly,cotreatment with FIIN-2 and CQ/3-MA also significantly increased the expression of the proapoptotic proteins Bax and cleaved Caspase-3.11.Combined with autophagy inhibitors augmented the anti-LUAD effect of FIIN-2 in vivoThe results of in vivo experiments showed that in A549 and A549/DDP cells-derived subcutaneous xenograft tumor models of nude mice,FIIN-2 treatment could inhibit the growth of tumor,and the tumor volume and weight were significantly reduced,while FIIN-2 combined with CQ exerted a more obvious tumor-inhibiting effect.IHC results showed that FIIN-2 could inhibit the expression of Ki67,and the effect of FIIN-2 combined with CQ was more obvious.TUNEL staining showed that the apoptosis rate of FIIN-2 group was significantly increased,and the combined CQ group further increased,accompanied by the upregulation of cleaved Caspase-3.Meanwhile,the combination of CQ further increased the inhibitory effect of FIIN-2 on p-FRS2.Additionally,FIIN-2 increased LC3 levels and decreased p62 abundance,and combined with CQ further increased LC3 levels.ConclusionsThe irreversible FGFR inhibitor FIIN-2 could exert anti-LUAD effect by inhibiting FRS2-AKT/ERK-mTORC1 signaling.Specifically,FIIN-2 could inhibit cell proliferation,colony formation and migration,and can induce the mitochondria-mediated apoptosis in A549 and A549/DDP cells.Furthermore,FIIN-2 could induce pro-survival autophagy in A549 and A549/DDP cells by inhibiting mTORC1 and further activating the class Ⅲ PI3K complex.More importantly,FIIN-2-induced apoptosis lasted longer than autophagy,thereby overcoming the role of protective autophagy,resulting in LUAD cell death.Therefore,combined autophagy inhibitors could enhance the cytotoxic effect of FIIN-2 in vitro and in vivo.And the anti-LUAD and autophagy-inducing effects of FIIN-2 on A549/DDP cells were significantly higher than those of A549 cells,which was related to its more obvious inhibition of AKT-mTORC1 signaling in A549/DDP cells. |
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